A potential for future improvements in the treatement of caridvacular disorders may be contributed by approaches as disclosed in this invention which directely interfere with the collagen and/or vWF factor induced platelet adhesion.
Several novel inhibitors which prevent platelet adhesion are monoclonal antibodies directed to vWF. It has as well been suggested that glycoprotein IIb/IIIa inhibitors may be beneficial in inhibiting platelet adhesion
Some of these inhibitors like the monoclonal Ab c7E3 have already been tested clinically while others like the KGD- and RGDF-inhibitors are still under study. However, the specificity of most of these new inhibitors is not very well studied thus the spectrum of side effects that will be induced by using this inhibitors is still open and deserves carefull examination.
A rich source for the screening of new compounds that interfere with collagen induced platelet adhesion is given in nature through blood-sucking animals. Several inhibitors have been isolated from nature as described in the literature: A 65 kD protein called Calin isolated from Hirudo medicinalis (U.S. Pat. No. 5,587,360, WO 92/07005) (Munro, R., et al., Blood Coagulation and Fibrinolysis, 1991, 2, 179–184) and a 16 kD (LAPP) protein isolated from the salivary glands of the leech Haementeria officinalis (U.S. Pat. No. 5,324,715). Both proteins have been described as aggregation inhibitors as tested in static assays of collagen dependent platelet aggregation.
Despite a proven in vitro activity LAPP failed to act in several well-established in vivo models (Schaffer L. W. et al.; Arterioscler. Thromb., 1993, 13, 1593–1601) and Connolly T. M. et al.; Thromb. Haemostas., 1993, 69, 589). The soft tick, Ornithodoros moubata, also contains an antiplatelet protein (Moubatin) which is active in preventing collagen-stimulated platelet aggregation (Waxman, L. et al.; J. Biol. Chem., 1993, 268, 5445–49). Another inhibitor of platelet aggregation from a blood-sucking bug was disclosed in WO 9309137 by Noeske-Jungblut C. et al. Smith et al. have isolated a 50 kDa protein from snake venom and a 19 kDa protein was isolated from a the saliva of Triatoma pallidipennis, a blood-sucking bug. The protein was found to contain a factor that specifically inhibits collagen-induced platelet aggregation. The 19 kDa protein named pallidipin inhibits collagen-mediated aggregation of platelets in plasma. No inhibition of aggregation stimulated by other effectors (ADP, thrombin, thromboxane A2 mimetic U46619, phorbol ester) was detected. Pallidipin had no effect on platelet adhesion to collagen but inhibited ATP release from platelets. It interacted reversibly with platelets and may share with collagen a common target on them. The precise mechanism of action and therapeutic benefit of this protein is under investigation. Gan et al. described Echistatin as an inhibitor binding to the fibrinogen receptor GP IIa/IIIb (J. Biol. Chem. 1988, 263, 19827–32).
Despite these exciting developments, the need continues to exist for supplying further anticoagulants and antithrombin which have increased efficacy in the inhibition of clot formation, vWF-induced platelet activation or endothelial cell activation and which may be used pharmaceutically and produced in commercially feasible quantities.
Since none of the known proteins described so far has developed into a compound with ideal therapeutic profile the inventors of the present invention decided to go ahead with a new screening strategy in order to detect more relevant proteins.